The crystal structures of nerve growth factor (NGF), transforming growth factor-beta (TGF-β), platelet-derived growth factor (PDGF), and human chorionic gonadotropin (hCG), from four separate growth factor families revealed that these proteins are structurally related as a superfamily and share a common overall topology (McDonald and Hendrickson (1993) Cell 73:421-424). These proteins have little sequence homology, but they all have an unusual arrangement of six cysteines linked to form a “cystine-knot” conformation. One example of a cysteine framework in these proteins consists of four cysteine residues with a cysteine spacing of Cys-Xaa-Xaa-Xaa-Cys (SEQ ID NO:1) and Cys-Xaa-Cys, important for a ring structure formed by eight amino acids. The two additional cysteines form a third disulfide bond that penetrates the ring structure, thus forming the cystine-knot. Other variations of the eight amino acid ring structure include a fourteen- and a sixteen amino acid-ringed cystine-knot that nonetheless, maintains the same overall topology of the superfamily.
The active forms of these cystine-knot proteins are dimers, either homo- or heterodimers (Sun and Davies (1995) Annu. Rev. Biophys. Biomol. Struct. 24:269-291). Because of their structure, there appears to be an intrinsic requirement for the cystine-knot proteins to form dimers. This extra level of organization increases the variety of structures built around this simple structural motif (Isaacs (1995) Curr. Opin. Struct. Biol. 5:391-395).
The glycoprotein hormone subgroup of cystine-knot proteins are classically considered to be a family of four proteins (chorionic gonadotropin, CG; follicle stimulating hormone, FSH; luteinizing hormone, LH; and thyroid stimulation hormone, TSH) distinguished by the following characteristics. All members of this subgroup are non-covalently associated heterodimers that contain an α-subunit, which is the product of a common gene and a distinct β-subunit, which confers biological specificity (Pierce and Parsons (1981) Annu. Rev. Biochem. 50:465-95). Structurally, both the glycoprotein hormone α- and β-subunits each consist of three β-sheets stabilized by hydrogen bonds and a cystine-knot; the subunits align in a head-to-tail manner with the heterodimers being stabilized by a disulfide “seatbelt” (Lapthorn, et al. (1994) Nature 369:455-61). Glycoprotein hormones are secreted from either the pituitary (FSH, LH and TSH) or the placenta (CG). Because of their respective roles in human reproduction, FSH, LH and CG are collectively termed gonadotropins.
To study the structure and biological function of cystine-knot proteins, these proteins have been purified using various methods.
Partially purified hCG has been used as starting material (Bahl (1969) J. Biol. Chem. 244:567-574; Birken, et al. (1988) Endocrinology 123:572-83; Canfield (1971) Recent Prog. Horm. Res. 27:121-64; Morgan, et al. (1974) Endocrinology 94:1601-6) and, while relatively simple, were only of use for purifying human urinary CG. Purification of gonadotropins from urine or culture media is considerably more complex, and has involved the use of organic or ammonium sulfate precipitation, and hydrophobic interaction chromatography and ion-exchange chromatography (Reisfeld and Hertz (1960) Biochem. Biophys. Acta 43:540-3; Gadkari, et al. (2003) Protein Expr. Purif. 32:175-84), or antisera (Jiang, et al. (2002) Mol. Hum. Reprod. 8:201-12; Manna, et al. (2002) Hum. Mol. Genet. 11:301-15). Another purification scheme for CG uses a combination of ammonium sulfate precipitation, hydrophobic interaction chromatography, and ion exchange chromatography (Gadkari, et al. (2003) supra).
Kawate and Menon ((1994) J. Biol. Chem. 269:30651-58); Zheng and Menon ((1989) Proc. Natl. Acad. Sci. USA 86:8294-8); and Munshi et al. ((2001) Eur. J. Biochem. 268:1631-1639) teach that hCG covalently binds to AFFI-GEL® 10 beads and may be used as a ligand in an affinity column for isolating the LH/hCG receptor.
U.S. Pat. No. 6,414,123 discloses a method for purifying FSH from a sample by applying the sample in a first buffer comprising a pH of less than about 7.5 to a dye affinity chromatography matrix comprising a dye ligand; washing out contaminants from the chromatography matrix with a second buffer comprising a pH of less than about 9.0; and eluting the FSH with a third buffer comprising less than about 0.8 M NaCl and a pH of less than 5.0. An additional purification step is taught which encompasses a hydrophobic solid phase chromatography step.
Needed in the art is a general method for substantially purifying members of the cystine-knot superfamily of proteins that takes advantage of the common structural motif of these proteins. The present invention meets this need in providing a rapid, efficient, two-step method for purifying members of the cystine-knot superfamily.